Increase of xanthan production by cloning xps genes into wild-type Xanthomonas campestris

Previously, genomic banks of Xanthomonas campestris were constructed in Escherichia coli, using mobilizable broad-host-range cosmids as the vectors. Following conjugal transfer, genes involved in the biosynthesis of xanthan polysaccharide (XPS) were cloned by the ability to restore the mucoid phenotype to the non-mucoid mutants. In this study, all clones were transferred into the wild-type strain Xc17 to evaluate the effects of the cloned genes on XPS production. Most clones showed no significant effect; however, two plasmids, pP2401 and pP2201, caused 10 and 15% yield increases, respectively, compared with that of controls. While it was not clear how pP2201 caused the yield increase, the effect of pP2401 seemed to result from elevated phosphomannose isomerase activity. Since XPS synthesis in X. campestris is a very efficient process, only relatively small increases are to be expected; an enhancement of productivity by 10-15% is important to the commercial production of xanthan.     

The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003bp, with 4471 protein-coding genes and 62 RNA genes. Comparative genomics showed that the genes required for the synthesis of xanthan and xanthan precursors were highly conserved among three sequenced X. campestris pv. campestris genomes, but differed noticeably when compared to the remaining four Xanthomonas genomes available. For the xanthan biosynthesis genes gumB and gumK earlier translational starts were proposed, while gumI and gumL turned out to be unique with no homologues beyond the Xanthomonas genomes sequenced. From the genomic data the biosynthesis pathways for the production of the exopolysaccharide xanthan could be elucidated. The first step of this process is the uptake of sugars serving as carbon and energy sources wherefore genes for 15 carbohydrate import systems could be identified. Metabolic pathways playing a role for xanthan biosynthesis could be deduced from the annotated genome. These reconstructed pathways concerned the storage and metabolization of the imported sugars. The recognized sugar utilization pathways included the Entner-Doudoroff and the pentose phosphate pathway as well as the Embden-Meyerhof pathway (glycolysis). The reconstruction indicated that the nucleotide sugar precursors for xanthan can be converted from intermediates of the pentose phosphate pathway, some of which are also intermediates of glycolysis or the Entner-Doudoroff pathway. Xanthan biosynthesis requires in particular the nucleotide sugars UDP-glucose, UDP-glucuronate, and GDP-mannose, from which xanthan repeat units are built under the control of the gum genes. The updated genome annotation data allowed reconsidering and refining the mechanistic model for xanthan biosynthesis.     

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.     

Genetic stability and xanthan gum production in Xanthomonas campestris pv. campestris NRRL B1459

A transposon (Tn5-SC) was constructed that can be used to quantify genetic deletions or amplifications. This transposon was used to evaluate the genomic stability of Xanthomonas campestris pv. campestris NRRL B1459 and we found that the genome of this bacterium is as stable as other Gram-negative bacteria or even more stable. Homologous recombination between plasmid sequences was determined in strain NRRL B1459 and was found to occur at a similar level to that reported for other Gram-negative bacteria. We report here that in X.c.c. NRRL B1459 there is no straightforward correlation between the occurrence of genetic rearrangements and frequency of homologous recombination. These data are discussed with respect to the reported instability of strain NRRL B1459 for xanthan gum production.     

Detection of Xanthomonas campestris mutants with increased xanthan production

Several mutants of Xanthomonas campestris showing increased viscosity and/or gum production were detected after UV treatment. Xanthan solutions of the different mutants showed different intrinsic viscosity values, and no relationship was found between pyruvate or acetate contents and viscosifying ability of the xanthan. The best performance mutant (M-11) was obtained using halo size around colonies in starch-agar plates as the detection criterion, and proved the usefulness of this indirect screen for improved xanthan producers. A pleiotropic effect of this mutation on growth rate and total cell growth was observed. Received 07 December 1995/ Accepted in revised form 14 November 1996     

Variations in xanthan production by antibiotic-resistant mutants of Xanthomonas campestris

Mutants resistant to different antibiotics (streptomycin, tetracycline, ampicillin and penicillin) were obtained from several strains of Xanthomonas campestris and evaluated for xanthan production. Most of the mutants showed alterations in their polysaccharide production, either increasing, decreasing or totally losing their polymer-production capacity. The existence of two types of antibiotic-resistance mechanisms for the assayed drugs is suggested: one that affects xanthan production and another that does not. Differences in outer-membrane protein patterns of mutants that were simultaneously altered in antibiotic resistance and xanthan production were found, in comparison with their parental strains. These findings suggest the existence of a genetic relationship between antibiotic-resistance mechanisms and xanthan production. Some of the mutants obtained showed significant increases in broth viscosity and xanthan concentration. These results suggest that resistance to streptomycin and ampicillin can be used to obtain improved strains in plate screening assays. Received: 8 January 1997 / Received revision: 13 June 1997 / Accepted: 4 July 1997     

Lipopolysaccharide biosynthesis in Xanthomonas campestris pv. campestris: a cluster of 15 genes is involved in the biosynthesis of the LPS O-antigen and the LPS core

As a result of mutational and DNA sequence analysis, a wxc gene cluster involved in the synthesis of the surface lipopolysaccharide (LPS) was identified in Xanthomonas campestris pv. campestris. This gene cluster comprises 15 genes. It was located on a cloned 35-kb fragment of chromosomal DNA, close, but not directly adjacent, to previously characterized genes for LPS biosynthesis. The G + C content of all but one of the wxc genes was atypically low for X. campestris pv. campestris, while the G + C distribution was uniform throughout the cluster. An SDS-PAGE analysis of mutant strains defective in various wxc genes confirmed that genes from this cluster were involved in LPS biosynthesis. The mutant phenotypes allowed the differentiation of three regions within the wxc cluster. Genes from wxc region 1 are necessary for the biosynthesis of the water-soluble LPS O-antigen. Analysis of DNA and deduced amino acid sequences led to the identification of two glycosyltransferases, two components of an ABC transport system, and a possible kinase among the seven putative proteins encoded by genes constituting wxc region 1. The two genes in wxc region 2 were similar to gmd and rmd, which direct the synthesis of the sugar nucleotide GDP-D-rhamnose. Mutations affecting wxc region 2 demonstrated its involvement in the formation of the LPS core. Genes from wxc region 3 showed similarities to genes that code for enzymes that modify nucleotide sugars, and to components of sugar translocation systems that have so far been rarely described in bacteria.     

Stimulation of xanthan production by Xanthomonas campestris using citric acid

The biosynthesis of xanthan by Xanthomonas campestris was found to be affected by the addition of citric acid in fed batch mode. Under oxygen-limiting conditions, the addition of up to 2.6 g citric acid per litre improved cell viability as well as increasing xanthan yield by up to 80%. Comparative xanthan formation profiles at different operating conditions indicate that at higher aeration (when there was no oxygen limitation), citric acid addition did not improve xanthan production.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey.

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Use of agricultural wastes for xanthan production by Xanthomonas campestris

Four different acid-hydrolyzed wastes, from melon, watermelon, cucumber and tomato were compared for xanthan production. Growth of Xanthomonas campestris, xanthan biosynthesis, kinetics and chemical composition were investigated. Both growth and xanthan production were dependent on the acid hydrolysate concentrations and available nitrogen. Melon acid hydrolyzed waste was the best substrate for xanthan production. Exopolysaccharide obtained throughout this study was compared to commercial xanthan, showing a very similar chemical composition. Acid hydrolyzed wastes are proposed as a new carbon source for xanthan production. Received 16 July 1998/ Accepted in revised form 8 October 1998     

Genetic construction of Xanthomonas campestris and xanthan gum production from whey

Summary Plasmids pUR291 and pNZ521 containing lacZ gene, maturation protein and proteinase P genes, were transferred into X. campestris either by conjugation or by transformation. Plasmid pNZ521 was also conjugally transferred into X. campestris XMT1 a transformant carrying plasmid pUR291. All the constructed strains were evaluated for xanthan gum production in either a medium of 50% whey or the same medium supplemented with 1.5% lactose or 1.5% glucose. Mixed cultures either with transconjugants or with transformants were tested for xanthan gum production as well.     

Growth and xanthan production of Xanthomonas campestris depending on the N-source concentration

Growth of X. campestris and production of xanthan were studied in several batch fermentations with different starting concentrations of N-source. The dependencies of growth, productivity and yields on initial N-source concentration were observed. The maximum yields in the course of cultivations were identified.List of Symbols Y _e economical yield coefficient - Y _P/sc product yield coefficient related to C-source - r _N specific growth rate related to cell number (h^–1) - Y _exp experimental yield coefficient - Y _T theoretical yield coefficient - c(Sn) concentration of N-source (mg/l) - c(Sc) concentration of C-source (g/l) - c(P) concentration of product (g/l) - N cell number (l^–1) - thermodynamic efficiency We are grateful to Mrs. B. Bhalová, PhD, for chemical analyses of the medium.     

Preservation of Xanthomonas campestris on agar slopes: effects on xanthan production

Xanthomonas campestris NRRL B-1459 and a variant E2, when preserved on agar slopes (transferred monthly) over 11 months did not deteriorate in their ability to produce xanthan in quantity and quality, as determined by culture in 500-ml baffled flasks. Variations between 8 and 14% (with respect to the average) in the final xanthan concentration were observed for the E2 and B-1459 strains, respectively. A wide range of final viscosities was obtained; these were consistent with the changes in gum concentration. Differences were more likely associated with differences in fermentation kinetics rather than being inherent to the strains. The rheological quality of both polysacharides was relatively constant throughout the time of culture maintenance. Preservation of these bacteria on agar slopes was an adequate method, in contrast to previous reports. In the period studied, strain E2 produced higher gum titres and slightly lower gum quality compared to strain B-1459. Correspondence to: E. Galindo     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Influence of nutrients on cell growth and xanthan production by Xanthomonas campestris

Xanthan production by Xanthomonas campestris was performed in a batch fermentation study with a view to achieving maximum yields. The factors influencing the production — mainly the nutrients, were investigated. Trace elements such as phosphate and magnesium at 6.0 g/l and 0.2 g/l enhanced the yields.Dr. M. Chellapandian is thankful to the Council of Scientific and Industrial Research, New Delhi for the award of Research Associateship.     

Effect of corn steep liquor on xanthan production by Xanthomonas campestris

Summary The addition of corn steep liquor (CSL) to batch cultures of Xanthomonas campestris using sucrose as carbon source stimulated cell growth rate, viscosity and xanthan production as compared to non-supplemented cultures. The addition of CSL to a basal medium at a dose of 1 g/l, increased xanthan production and viscosity by 22% and 44% respectively. CSL also shortened the cultivation time and promoted a more efficient sucrose utilization for polymer synthesis. After 72 h of incubation the xanthan yield per sucrose consumed in the CSL-amended culture was 0.63 g/g, this is, 15% higher than without CSL addition. At higher doses of CSL cell growth rate was also increased but not polymer production.     

Genetic engineering of polysaccharide structure: production of variants of xanthan gum in Xanthomonas campestris.

Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. Xanthan has wide commercial application as a viscosifier of aqueous solutions. Previously, through genetic engineering, a set of mutants defective in the xanthan biosynthetic pathway has been obtained. Certain mutants were shown to synthesize and polymerize structural variants of the xanthan repeating unit and thus produce "variant xanthans". Initial studies of solution viscosities of these polymers, presented here, indicate that the variants have rheological properties similar to, but not identical with, xanthan. These results indicate that acetylation and pyruvylation can affect the viscometric properties of xanthan. Specifically, the presence of pyruvate increases viscosity, whereas acetate decreases viscosity. In addition, the elimination of sugar residues from xanthan side chains also has a major effect on viscosity. Compared to wild-type xanthan, polymer lacking the terminal mannose (polytetramer) is a poor viscosifier. In contrast, polymer lacking both the terminal mannose and glucuronic acid (polytrimer) is a superior viscosifier, on a weight basis. There is a negative effect of acetylation on the viscosity of polytetramer xanthan, but there is seemingly no effect of acetylation on polytrimer xanthan viscosity. The further study of these materials should provide insight into the relationship between xanthan structure and rheological behavior.     

Expression of the gum operon directing xanthan biosynthesis in Xanthomonas campestris and its regulation in planta

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of beta-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.